Base-Catalyzed Phenol-Mannich Condensation of Preformed Cesium Iminodiacetate. The Direct Synthesis of Calcein Blue AM and Related Acyloxymethyl Esters.

A rapid and highly practical one-flask procedure for the positionally selective preparation of (acyloxy)methyl N-(2-hydroxybenzyl)iminodiacetate and related diesters from iminodiacetic acid and phenols is described. The key to this multicomponent phenol-Mannich condensation resides in the use of cesium iminodiacetate as the reaction partner. This protocol has been applied in an unusually direct synthesis of the intracellular fluorescent dye Calcein blue AM, for which scant experimental and spectroscopic data are presently available.

Stepwise PEG synthesis featuring deprotection and coupling in one pot.

The stepwise synthesis of monodisperse polyethylene glycols (PEGs) and their derivatives usually involves using an acid-labile protecting group such as DMTr and coupling the two PEG moieties together under basic Williamson ether formation conditions. Using this approach, each elongation of PEG is achieved in three steps - deprotection, deprotonation and coupling - in two pots. Here, we report a more convenient approach for PEG synthesis featuring the use of a base-labile protecting group such as the phenethyl group. Using this approach, each elongation of PEG can be achieved in two steps - deprotection and coupling - in only one pot. The deprotonation step, and the isolation and purification of the intermediate product after deprotection using existing approaches are no longer needed when the one-pot approach is used. Because the stepwise PEG synthesis usually requires multiple PEG elongation cycles, the new PEG synthesis method is expected to significantly lower PEG synthesis cost.

A FRET-Based Near-Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria.

We report a near-infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near-infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18-fold and the fluorescence of the rhodamine acceptor decreased moderately by 45 % under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.

Near-Infrared Hybrid Rhodol Dyes with Spiropyran Switches for Sensitive Ratiometric Sensing of pH Changes in Mitochondria and Drosophila melanogaster First-Instar Larvae.

Near-infrared hybrid rhodol dyes (probes A and B) for sensitive ratiometric visualization of pH changes were prepared by incorporating hemicyanine dyes into traditional rhodol dyes. This approach was based on π-conjugation changes involving a rhodol hydroxyl group as a spiropyran switch upon pH changes. Electronic spectra of probes A-2 and B-2 contain sharp absorption peaks at 535 nm and fluorescence peaks at 558 nm with similar π-conjugation and a closed spiropyran form at a basic pH of 10.2. However, acidic pH conditions break down the hemiaminal ether groups, leading to indolenium moieties and significantly extending the π-conjugation within the rhodol fluorophores, resulting in additional near-infrared emissions for probes A-1 and B-1. As a result, probes A and B exhibit gradual decreases of the absorption peaks at 535 nm and gradual increases in absorption peaks at 609 and 622 nm upon transition from basic to acidic pH, respectively. Both probes display ratiometric fluorescence sensing responses to pH downgrades from 10.2 to 3.6 with visible fluorescence decreases at 558 nm, as well as corresponding increases of the near-infrared fluorescence peaks at 688 and 698 nm, respectively. They exhibit fast, sensitive, and selective fluorescence responses with clearly defined ratiometric features to pH changes and show low cytotoxicity and excellent cell permeability. Our probes were successfully applied to ratiometrically detect pH changes in mitochondria, D. melanogaster first-instar larvae, and to visualize the mitophagy process caused by either cell nutrient starvation or drug treatment.

A Near-Infrared Fluorescent Probe Based on a FRET Rhodamine Donor Linked to a Cyanine Acceptor for Sensitive Detection of Intracellular pH Alternations.

A fluorescence resonance energy transfer (FRET)-based near-infrared fluorescent probe (B⁺) for double-checked sensitive detection of intracellular pH changes has been synthesized by binding a near-infrared rhodamine donor to a near-infrared cyanine acceptor through robust C-N bonds via a nucleophilic substitution reaction. To demonstrate the double-checked advantages of probe B⁺, a near-infrared probe (A) was also prepared by modification of a near-infrared rhodamine dye with ethylenediamine to produce a closed spirolactam residue. Under basic conditions, probe B⁺ shows only weak fluorescence from the cyanine acceptor while probe A displays nonfluorescence due to retention of the closed spirolactam form of the rhodamine moiety. Upon decrease in solution pH level, probe B⁺ exhibits a gradual fluorescence increase from rhodamine and cyanine constituents at 623 nm and 743 nm respectively, whereas probe A displays fluorescence increase at 623 nm on the rhodamine moiety as acidic conditions leads to the rupture of the probe spirolactam rings. Probes A and B⁺ have successfully been used to monitor intracellular pH alternations and possess pKa values of 5.15 and 7.80, respectively.